Correlation of HBsAg and HBV DNA
Michael Chudy, Paul-Ehrlich-Institut SoGAT XVIII, Bethesda MD 24-25 May 2005
PEI
HBsAg and HBV DNA
- Most sensitive HBsAg assay detects 0.01 ng/ml (PEI HBsAg standard; ad) - HBsAg cut-off correspond to HBV DNA
• 114 IU/ml (WHO DNA standard) • 461 copies/ml (five SC panels; ZeptoMetrix)
Good correlation to published data from Biswas et al.; Transfusion 43:788-798 (2003)
Virus and HBsAg Particles
HBsAg reactive polypeptides
• Most sensitive ELISA detects 10 pg HBsAg/ml • Virion-HBsAg – One HBV particle: 20 ag HBsAg* – 10 pg HBsAg correspond to about 500,000 HBV particles • 500 particles/ml (HBsAg cutoff of SC panels) vs. 500,000 HBV particles/ml • Viral surface antigen reactive polypeptides – Virion envelope – Incomplete viral forms • Excessive incomplete viral forms are present (about 1:1,000)
*Average numbers of molecules of LHBs, MHBs, and SHBs: 30, 30, and 350, respectively; Calculation correlate well with data presented by Prof. Gerlich (EPFA meeting 2002)
Correlation of HBsAg and HBV DNA
Is that true for all geno-/subtypes, stages … of HBV infection? Can HBV NAT replace the HBsAg testing in blood donors?
HBsAg and HBV DNA
• HBV DNA: reverse transcription from 3.35 kb pre-genomic mRNA • HBsAg forms translated from
LHBs MHBs/SHBs 2.4 kb mRNA 2.1 kb mRNA
• Level of regulation
- Transcription - Post-transcription
e.g. Transport of unspliced mRNAs from nucleus to cytoplasm
- Cellular factors
Ratio of total HBsAg to Virion-HBsAg at different HBV stages
• Early period of HBV infection
– Genotype A – Genotype G
• Chronic HBV infection
Total HBsAg/Virion-HBsAg ratio in samples of SC panel PHM911 (BBI)#
1000000 1
1:490
100000
HBV DNA (geq/ml) HBsAg (ng/ml)
10000 1000 100 10 1
1:200
1:1,100
21 d
1:730
1:2,200
HBV DNA HBsAg
#data
50
60
70
80
90
0,1 100
Days after 1st Bleeding
performed in 1995
HBV genotype G
– Only few infections are known (all coinfections with genotype A) – First report of HBV infection and transmission based exclusively on genotype G (2003 in Germany) – No escape variant (subtype adw2) – HBeg minus variant
Studies were performed in cooperation with the Institute of Transfusion Medicine and Immunohematology, GRC, JW Goethe University, Frankfurt (WK Roth and co-workers)
HBV transmission exclusively by genotype G-virus
Sample Ctr Ctr Ctr PP TP (3) PP TP (2) TP (1) PP PP PP R1 - (1, double) R2 - (2+3) R3 - (2) Date HBsAg 2003-06-18 neg 2003-06-11 neg 2003-06-04 pos 2003-05-28 pos 2003-05-19 neg 2003-05-14 neg 2003-05-09 neg 2003-04-30 neg 2003-04-24 neg 2003-04-17 neg 2003-04-10 neg 2003-06-19 neg 2003-07-01 neg 2003-06-11 neg 2003-06-23 neg 2003-06-30 neg 2003-09-23 pos 2003-06-13 neg 2003-06-19 neg 2003-07-10 neg 2003-07-21 neg 2003-07-31 neg 2003-08-08 neg 2003-08-22 neg (s/co) 2003-09-01 neg (s/co) 2003-09-09 neg (s/co) **COBAS TaqMan HBV Test SD-NAT (cps/mL) neg 20 pos (qual) 8210* 6.258 4.000 855 295 141 48 neg neg neg 52 579 2.670 >10.000 69 229 3.900 12.200 5.480 6.490 16.120 2.350 380 SD-NAT** (cps/mL) anti-HBc pos neg neg 2.872 anti-HBs pos (18 IU/ml) pos (36 IU/ml) neg
Donor
80 <35 pos (IgM neg) neg 779 1.573 9.273.200 neg neg neg neg neg neg neg pos pos pos (135 IU/ml) pos (164 IU/ml)
Recipient
neg neg neg neg neg neg neg neg
R4 - (3)
10.741 16.296 9.062 13.890 1.152
*Pool-PCR pos
Virion-HBsAg/total HBsAg ratio in genotype G
Sample from R3 (2003-09-23) with about 9 Mio cps/ml (positive for HBsAg, negative for anti-HBc, HBeAg/antiHBe) - Titration in Prism HBsAg test - Virus concentration at HBsAg cut-off: 24,000 copies/ml - Ratio of virion-HBsAg to total HBsAg of 1:20 - vs. 1:1,000 and more in genotype A samples of early HBV infection - Significant decrease of excessive HBsAg in infection with genotype G
HBsAg and HBV DNA in chronic HBV infection
• Lack of correlation between HBsAg and HBV DNA in HBsAg/anti-HBc positive tested samples (Kuhns et al. 2004, Transfusion 44,1332-1339)
HBsAg and HBV DNA in chronic HBV carriers
• Investigation of 106 chronic carriers tested positive for HBsAg, anti-HBc, and anti-HBe • Only 84% (89/106) HBV NAT reactive with the Procleix Ultrio Assay (Gen-Probe)
– – – – – s/co s/co s/co s/co s/co >20 15-20 10-15 5-10 <5 5x 65x 9x 7x 3x
• No correlation to s/co values of HBsAg test (Prism)
17 HBsAg positive/HBV DNA negative samples of chronic HBV carriers
Sample ID#
Prism HBsAg (s/co)
1:10 1:100 1:1.000
Procleix Ultrio Assay
s/co analyte
14
15 22
0,58
7,39 99,11
0,42
1,01 0,80
0,39
0,45 0,50
0,03
0,07 0,04
63
72
178,51
218,48
1,57
14,33
0,47
1,59
0,04
0,30
74
76 96 111 112
2,08
27,30 170,96 0,62 51,98
0,51
2,00 8,74 0,43 3,89
0,43
0,64 1,14 0,40 0,80
0,06
0,04 0,05 0,06 0,04
120
122
2,12
0,45
0,62
0,45
0,44
0,45
0,05
0,08
141
161 164 173 184
37,33
228,31 13,89 1,08 0,44
3,56
58,59 2,16 0,60 0,43
0,77
5,76 0,61 0,43 0,45
0,11
0,15 0,08 0,33 0,06
Summary: Correlation of HBsAg and HBV DNA
• Virion-HBsAg of one particle (20 ag) correspond to one copy of HBV DNA • Significant excess of HBsAg particles in the early period of HBV infection (genotype A) • Variable ratio of Virion-HBsAg to total HBsAg in the course of infection (genotype A) • Genotype G infection shows a significant decrease in synthesis of incomplete viral forms • Lack of correlation between HBsAg and HBV DNA in chronic infection • HBsAg carrier (integrative phase) can be nonreactive by HBV NAT • Results indicate caution in any consideration of dropping HBsAg screening